Much of the progress in transplantation can be attributed to new immunosuppressive drugs whose use is a life-long requirement. Chronic immunosuppression has many complications (infections, neoplasms, drug and metabolic toxicity). A well-tolerated method to induce allograft tolerance remains the ultimate goal of clinical transplantation. We have utilized non-radiation based lymphoablation with antilymphocyte serum (ALS), rapamycin (sirolimus) and donor bone marrow (BM) to induce indefinite tolerance to fully MHC incompatible murine skin allografts. Tolerance is associated with multilineage chimerism and clonal deletion. The chimeric donor cells are B cells, macrophageslmonocytes (no T cells are identified). T and B cells are not required in the donor BM for tolerance induction, but class II expressing BM cells are absolutely required. In Specific Aim 1 we will identify toferogenic BM cell subsets by deleting or enriching/purifying specific BM class II antigen positive cells and hematopoietic stem cells (HSCs). We will identify additional sources of tolerogenic BM derived cells using cytokine mobilized peripheral blood stem cells (PBSCs) and in vitro culture generated BM dendritic cells (DCs). Experiments will be performed to determine if BM DC's require co-stimulating capacity to induce tolerance. In Specific Aim 2 the role of peripheral tolerance mechanisms (activation induced cell death and/or suppressor cell cytokines) will be studied. The relationship of T cell apoptosis to tolerance will be determined by defining the importance of intact apoptotic pathways using knockout recipients, by correlating the extent of T cell apoptosis in lymphoid tissue with the degree and duration of tolerance as well as quantitating the proliferative capacity and susceptibility to apoptosis in tolerant and non-tolerant recipients. The role of suppressor cytokines will be investigated by determining the proliferative capacity and production of tolerogenic Th-2 cytokines as well as the capacity to induce tolerance in IL-4 and IL-10 knockout recipients. The ease of application, the long clinical experience with ALS, the availability of rapamycin, and the frequent utilization of BM transplantation as well as lack of radiation requirements make this method of tolerance induction very attractive for clinical application.